The present invention relates generally to methods of separating nucleic acids from cellular debris, such as proteins, in a biological sample. Specifically, the invention relates to the use of electrophoresis at a low pH to separate nucleic acids from substances carrying a net positive charge.
Technology to detect minute quantities of nucleic acids has advanced rapidly over the last two decades including the development of highly sophisticated amplification techniques such as polymerase chain reaction (PCR). Researchers have readily recognized the value of such technologies to detect diseases and genetic features in human or animal test specimens.
PCR is a significant advance in the art to allow detection of very small concentrations of a targeted nucleic acid. The details of PCR are described, for example, in U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis) and U.S. Pat. No. 4,965,188 (Mullis et al), although there is a rapidly expanding volume of literature in this field. Without going into extensive detail, PCR involves hybridizing primers to the strands of a targeted nucleic acid (considered xe2x80x9ctemplatesxe2x80x9d) in the presence of a polymerization agent (such as DNA polymerase) and deoxyribonucleoside triphosphates under the appropriate conditions. The result is the formation of primer extension products along the templates, the products having added thereto nucleotides which are complementary to the templates.
Once the primer extension products are denatured, and one copy of the templates has been prepared, the cycle of priming, extending and denaturation can be carried out as many times as desired to provide an exponential increase in the amount of nucleic acid which has the same sequence as the target nucleic acid. In effect, the target nucleic acid is duplicated (or xe2x80x9camplifiedxe2x80x9d) many times so that it is more easily detected.
In order to effectively amplify and detect a target nucleic acid or to clone or sequence a target nucleic acid, it is frequently necessary to isolate or separate the nucleic acid from a mixture of other interfering biomolecules. (Moore D., 1997. Preparation and Analysis of DNA. Unit 2.2 In Ausubel et al. (ed.), Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York.)
Presently, several different procedures are used to remove proteins and other impurities from nucleic acid preparations. Traditionally, biological samples were digested with a protease, and impurities removed from the nucleic acids by organic extraction (Moore D., Current Protocols in Molecular Biology). This method, however, has several recognized disadvantages including using hazardous organic solvents and requiring several transfers of aqueous phase to fresh tubes, which is tedious, labor intensive, and adds to the risk of cross contaminating samples.
Purification of nucleic acids by adsorption to glass in a chaotropic salt has become popular more recently (Boom et al., 1990, J. Clinical Microbiol. 28:495-503). However, this separation method also suffers from several disadvantages including using glass that has a very low binding capacity, employing chaotropic salts, and being tedious and time consuming because the glass-nucleic acid complex must be washed several times and the wash solution removed.
Polymer capture, an ion exchange procedure, to purify DNA has also been employed to isolate nucleic acids (U.S. Pat. Nos. 5,582,988, 5,434,270, and 5,523,368). Unfortunately, such procedures are not particularly suitable for RNA purification under the conditions currently practiced because RNA would be degraded both during capture and release. Under optimal polymer-nucleic acid capture conditions ribonuclease activity would be high resulting in degradation of the RNA and the high pH needed to release nucleic acids from the polymer would result in chemical hydrolysis.
Electrophoretic separation is an appealing technique because such procedures can be designed to avoid hazardous substances, high pH, and tedious manipulations. In addition, electrophoretic separation is readily adaptable to automated formats. Although electrophoresis is most often used on an analytical scale, many small-scale preparative procedures have been developed as well (Andrews, A. T., 1986, Electrophoresis: Theory and Techniques, and Biochemical and Clinical Applications, 2nd edition. Clarendon Press, Oxford, England). Procedures have also been reported that separate DNA from humic materials and other impurities that inhibit PCR by electrophoresis on polyvinylpyrrolidone-agarose gels (Herrick et al., 1993, Appl. Environ. Microbiol. 59:687-694 and Young et al., 1993, Appl. Environ Microbiol., 59:1972-1974). In addition, Sheldon and co-workers developed a device for electrophoretic purification of nucleic acids. Cells or blood samples are lysed with a protease and the lysate is loaded into the device. Nucleic acids are separated from impurities by electrophoresis through a polymer layer and are retained in a collection chamber by a molecular weight cut-off membrane, while degraded proteins and other low molecular weight substances pass through the membrane (Sheldon E. L., 1997. Electronic Sample Handling. Presented at International Business Preparation Workshop. San Diego, Calif., Jun. 9, 1997).
Although most electrophoretic separations are run at a pH close to neutral, including those examples mentioned above, electrophoresis at low pH is sometimes advantageous. For example, some macromolecules separate more efficiently at low pH. Mixtures of nucleotides or low molecular weight polynucleotides separate better at low pH because the charge on nucleotides varies from negative 1 to 0 between pH 2 and 5, while most have the same charge (minus 2) between pH 6 and 8 (Smith, J. D., 1976, Methods Enzymol. 12:350-361). Similarly, with isoelectric focusing, acidic proteins are isolated at their pKa in a pH gradient (Andrews, A. T., 1986). In addition, some structures are more stable at low pH. Terwillinger and Clarke reported that acidic conditions (pH 2.5) help minimize hydrolysis of protein methyl esters during electrophoresis (Terwillinger et al., 1981, J. Biol. Chem. 256:3067-75). Similarly, triple helix structures of B-DNA are stabilized by mild acid conditions (pH 4.5) (Mirkin et al., 1987, J. Biol. Chem. 234:1512-16).
Accordingly, it would be desirable and advantageous to be able to use low pH conditions for preparative electrophoresis of nucleic acids, especially from protein-rich sources such as blood or plasma. Nucleases, especially ribonucleases, which are ubiquitous and will degrade nucleic acids during electrophoresis, are inactivated at low pH (Kalnitsky et al., 1959, J. Biol. Chem. 234:1512-16). Furthermore, most nucleic acids and proteins will have opposite charges under acid conditions, and therefore, will migrate in opposite directions in an electric field. At pH 2, nucleic acids will still be negatively charged, because the pKa values of the primary phosphate groups are less than 2 while those of the amine groups are between 2 and 5 (Smith, J. D., 1976). On the other hand, most proteins will be fully protonated, and therefore, positively charged because the pKa""s for all the amine and most carboxyl groups of proteins are much greater than 2.
Accordingly, the present invention overcomes the above-noted problems and provides a needed means for separating nucleic acids from substances that carry a net positive charge at low pH by electrophoresis under acid conditions. Electrophoresis at low pH also overcomes many of the problems with current methods for nucleic acid purification. As mentioned above, nucleases are less active at low pH, so the nucleic acids would be more stable than at neutral pH. Furthermore, once samples are loaded, electrophoresis is a hands-off method. Finally, no hazardous materials are needed.
Various other objects and advantages of the present invention will be apparent from the detail description of the invention.